Amphiphilic molecules, for example lipids, are known to aggregate in solution to form membrane structures which may be monolayers, micelles or liposomes. These structures have been shown to have semi-permeable properties which, in some examples, show selective passage of molecules (eg ions, ligands, antagonists, agonists). The selective permeability relates to the chemistry of the lipids used in the construction of the membrane structures. It is also known that these synthetic membranes may incorporate larger molecules such as polypeptides or proteins which function to, for example, facilitate the transport of molecules (for example, ion-channels which facilitate the transport of ions across membranes are referred to as ionophores), act as receptors for ligands, form pores through which polypeptides may be translocated (eg nuclear pore forming structures, mitochondrial protein import structures). These membrane polypeptides are typically referred to as membrane proteins.
Since membrane proteins have the intrinsic ability to assemble at interfaces in combination with other amphiphiles, they are suited to the construction of biomimetic surfaces for use in biocompatible devices and biosensors. Biosensors incorporating membrane polypeptides have many potential applications including, by example and not by way of limitation, ligand based biosensors for clinical diagnostics; the detection of contaminants in water and environment; memory devices; screening devices for pharmaceutical applications; the provision of biologically functionalised surfaces; binding sites for, and this sensors for, small molecules such as drugs, pesticides, molecules required to be analysed during process control (i.e. food stuffs, fermenter products, chemicals); larger molecules such as proteins for research screening (eg array technology) or diagnostics (cancer markers, infectious disease markers, hormones); nucleic acids; carbohydrate polymers; cells such as pathogenic bacteria; eukaryotic cells such as cancer cells and small single or multicellular organisms especially parasites. Moreover, a biosensor may contain membrane polypeptides combined with other components independently attached to the surface of the biosensor which themselves act as specific binding sites and for which the membrane polypeptide provides a stable non-denaturing surface and/or a ion-channel dependent sensor function. Biosensors can be employed in high throughput screening for pharmaceutical applications using ion channel modulation or the other methods describe above.
Biosensors can provide an inert, stable, biologically compatible assembly of biologically functionalised surfaces including peptides, nucleic acids; proteins and other large molecules. These structures can be used in screening and biosensor systems. They may also be used to create surfaces compatible with cell culture or implantation in living tissue.
Additionally, enzymes may be engineered into the membrane polypeptides or co-assembled with the membrane polypeptides such that they can be functionally and precisely assembled at surfaces. This could have applications in bioreactor systems where catalytic surfaces are required or sensor systems where the enzymatic product is more easily detected than the substrate. The enzyme plus membrane polypeptide could be specifically printed or applied such that 1, 2 or even 3 dimensional spatial arrangements can be defined. If combined with flow systems this may allow for sequential enzymatic synthesis/degradation along a small scale bioreactor.
It will be apparent to the skilled artisan that this technology also has broad applicability to the provision of surfaces which allow the linking of membrane polypeptides to a substrate in a particular orientation and density which allow these surfaces to function as binding surfaces for a range of molecules the interaction of which can be monitored by established methods. For example, optical methods such as surface plasmon resonance; fluorescence; ellipsometry; or electrical methods such as spectroscopy impedance, cyclic voltametry, conductance measurement. Membrane polypeptides could be used in biosensors in devices to sense physical signals such as voltage, pressure or temperature.
The modifiable permeability of the biosensor could be used to allow the controlled release of molecules across the surface from a reservoir below the layer. A layer composed of thiolipids and membrane polypeptides could also be used to trap molecules such as drugs in a reservoir. A biological signal such as pH, protease activity or ligand binding could trigger the release of the drag. One example could be microbeads targeted to specific tissues which release drugs through the membrane polypeptide channel when they bind to or enter the target cell. The membrane polypeptide could also be engineered to carry peptide sequences which by binding to specific cellular receptors would target the microbead to specific tissues. This could be in addition to or separate from the drug release function.
Ionophores are polypeptides or protein structures with a tertiary and in many cases quaternary structure forming pores embedded in cell membranes. Ionophores function to control the flow of ionic currents in response to either electrical excitation (referred to as voltage gating) or the presence of stimulatory ligands, for example neurotransmitters (referred to as ligand gating).
An example of a group of functionally related membrane proteins is the porins which is a sub-group of the ionophores. Other groups include GPCRs, pentameric ligand gated channels, ABC transporters etc.
The signal transducing properties of ion-channels, combined with their high sensitivity and their minimal size, has led to the development of biosensors based on synthetic membrane structures incorporating ion-channel proteins. Although there is no difficulty in manufacturing these membranes incorporating ion-channels they are often expensive to obtain or are fragile and unstable.
Prior art biosensors (see U.S. Pat. No. 5,234,566; U.S. Pat. No. 5,736,342; U.S. Pat. No. 5,516,890; WO9725616) are based on the tethering of a biolayer to the surface of a substrate in such a way that a layer is left attached to the substrate (eg by the use of thio-lipids). These biolayers serve as a substrate into which ion-channel forming polypeptides can be inserted during or after formation to provide a functional biological transducer. A problem associated with the synthetic membranes of this type is that when used with large integral membrane proteins of biological origin they lack durability, are expensive to manufacture and protein density and orientation is poorly controlled. In a particular case the device of Cornell (Cornell et al., 1997) uses a fluid lipid bilayer composed of half membrane spanning lipids and membrane spanning lipids which is tethered to the surface of thiolipids. The membrane allows for ease of diffusion in the plane of the bilayer and this is exploited by the use of gramicidin peptides. These synthetic half membrane spanning ionophore peptides only allow ion flow when a dimer is formed spanning the whole membrane. Generally one monomer is tethered to the substrate and the upper monomer is connected to a receptor molecule. Binding to the receptor alters the amount of conducting dimers present and results in a conductance change. This allows for biosensors of high sensitivity to be manufactured.